Interest in the insolubilisation of enzymes is well-known and an assessment of the methods of preparing enzymes fixed on an insoluble support has been described by several authors, particularly I. H. Silman and E. Katchalski, Ann. Rev. Biochem. (1966) 35, 873, Guilbault (1970) Anal. Chem. 425 334, R. and P. Cautrecasas and collaborators (1971) Ann. Rev. Biochem. 259.
The insolubilisation of enzymes has been carried out by four main types of methods. Firstly, insolubilisation can be achieved by physical adsorption of the enzyme on an inert support or on ion exchanger resins. However, with adsorption methods, there is a permanent danger of desorption of the enzyme occurring and the use of this method remains limited. The ion exchange method was usefully employed for adsorbing an amylocylase on dimethyl aminoethyl cellulose for the first time in 1966. Bernfeld and Wan (Science 142, 678, 1963) were the first to describe the second method for the insolubilisation of enzymes, in which a gel is incorporated in the pores of an ion exchange resin.
Thirdly, fixation of active proteins by covalent bonding on a water-insoluble support is the method which has been most widely used. The enzymatic derivatives which are obtained by this method generally present remarkable properties of resistance to the different denaturation factors. The fixation of the enzyme is effected by the intermediary of functional groups of the enzyme molecule which do not have any effect on the active site. In this type of insolubilisation, it is possible to distinguish between fixation on a previously activated support and fixation by the intervention of a polyfunctional agent. In fixation on an activated support, a functional group of the support is made sufficiently reactive to react with the enzyme. E. Brown (1970), Tetrahedron 25, 2139; Axen (1967) Nature 214, 1302; Porath (1967) Nature 215, 1491; P. Monsan (1971) C. R. Acad. Sci. Paris, 273, pages 33-36 series and French Patent Specification No. 2,133,370 have described the main methods which are based on this principle. In fixation by means of a polyfunctional agent, the latter is first of all fixed on the support and then the enzyme is brought into contact therewith. Examples of polyfunctional agents are given by Kay (1967), Nature 216, 514; H. H. Weetall (1969) Biochim. Biophys. acta 185, 464; and Neurath (1970) FEBS letters 8, 5, 253.
Among the insoluble supports which have been investigated in these different methods, cellulose (a polysaccharide) and its derivatives have frequently been used. Micheel and Envers (Makromol. Chem. 3, 200, 1949) were the first to use Curtins' method for activating the cellulose with an azide and fixing enzymes thereon. Concurrently with this technique of activating the cellulose and its derivatives, there is known the bromoacetyl method of Jagendorpf (Biochem. Biophys. Acta 78, 516, 1963), this implying the formation of a diazo compound as described by Campbell et al (Proc. Nat. Sci. U.S. 37, 575, 1951), that of the carbodiimide described by Weetall (Anal. Biochem. 14, 159-162, 1966) and that of the cyanogen halides described by Axen et al (Nature, 214, 1302-1304).
Among the enzymes which may be fixed by these methods are peroxydases, chymotripsin, trypsin, polynucleotide-phosphorylase, a .beta.-galactosidase, a lactate dehydrogenase and bromolane.
The fourth method of insolubulisation is characterised by the crosslinking, by covalent bonding, of an enzyme by means of bifunctional agents. This method consists in bonding together several molecules of enzyme (free or adsorbed) by means of a polyfunctional reagent, using one of the techniques published in the works of Silman and Katchalski, Ann. Rev. Biochem. (1966) 35 873; Haynes, (1969) Biochem. Biophys. res. Commun. 36, 235; Selegny (1968) C. R. Acad. Sci. 266, 1431; and French Patent Specification No. 1,604,982.
The methods of chemically fixing enzymes present disadvantages. In particular, the yields are low, the production costs are high and the enzymes are often denatured during the fixation phase.